The overall goal of this project is to examine in a representative higher primate, the rhesus monkey, the mechanisms that underlie the postnatal proliferation of undifferentiated stem spermatogonia and their niches, and that therefore determine the spermatogenic ceiling of the adult primate testis. Three Specific Aims will be addressed. Aim 1 will determine the spatial and temporal characteristics underlying the approximately 300-400 fold mitotic proliferation in the number of undifferentiated "stem" spermatogonia (A spermatogonia) from birth to adulthood in the monkey;Aim 2 will characterize the functional and molecular properties of undifferentiated "stem" spermatogonia in the testis of the juvenile monkey;and Aim 3 will establish the endocrine determinants of the pubertal expansion in Sertoli cell number, and presumably therefore that of spermatogonial stem cell niches. These aims will be addressed by combining classical morphometric approaches for quantitating cell numbers, with immunocytochemical analysis of S-phase and apoptotic cells in cross sections. Whole mounts and confocal analysis will be used to examine the spatial relationship between spermatogonial stem cell and Sertoli cell proliferation during prepubertal development. Spermatogonia will be isolated from the juvenile testis using a micromanipulator after digestion of seminiferous cords and their stem cell properties studied using the transplantation assay developed by Brinster and his colleagues, and "sternness" will be correlated to gene expression using RT-PCR and microarray analyses. Gene expression of A spermatogonial subtypes will also be studied in situ after cell capture with laser capture microdissection. Finally recombinant homologous gonadotropins and an androgen receptor antagonist will be employed to study the role of LH and FSH in stimulating spermatogonial stem cell niche (Sertoli cell) expansion during puberty. Successful completion of the project will provide the only systematic data on the ontogeny of the cell types dictating spermatogenic ceiling in the monkey and will provide the first information on the nature of male germline stem cells in the primate. This fundamental biological information will lead to a better understanding of male infertility and hopefully to improvement in reproductive health.